PacBio Reads subreads and scraps files

Question

[bio_d]

I have a PacBio library that I have converted from bam to fastq.gz files. Now when I hope to use them to correct, trim and assemble I cannot figure out whether I should use both the subreads and scrap files or only the former. The rationale for leaving out the later is Subreads because of the description provided in SMRT Link User Guide (from PacBio website). It says "Subreads that contain information such as double-adapter inserts or single-molecule artifacts are not used in secondary analysis, and are excluded from this file and placed in scraps.bam."

So if I don't use in assembler ( e.g. CANU) or PacBio Correction Tool (e.g. LoRDEC), am I doing it correctly or should I use both subreads and scraps files. Please let me know if I am doing it wrong.

Thanks in advance.

Answer 1

[tjduncan]

For assembly you would only need the "Subreads.bam".

You would only need the scraps.bam if you had multiplexed samples in your data-set that needed to have their barcodes identified to be de-multiplexed via CCS.

See: Brief primer and lexicon for PacBio SMRT sequencing for more info on file formats.

"Unaligned BAM files representing the subreads will be produced natively by the PacBio instrument. The subreads.bam will be the starting point for secondary analysis. In addition, the scraps arising from cutting out adapter and barcode sequences will be retained in a scraps.bam file, to enable reconstruction of HQ regions of the ZMW reads, in case the customer needs to rerun barcode finding with a different option."

[bio_d]

Thank you for the explanation and the link.

Answer 2

[GenoMax]

See this comment from Dr. Richard Hall (he is with PacBio) over at SeqAnswers.

[bio_d]

Thank you very much.